1. The roles of cyclin-dependent kinases (Cdks) in regulation of transcription and cell cycle Dalibor Blazek CEITEC-MU

2. Cyclin-dependent kinases (Cdks) Protein comlexes that compose of 1) Kinase subunit 2) Cyclin subunit Serine-threonine kinases-regulate function of proteins by phosphorylation of either Serine (S) or Threonine (T) Both subunits needed for the kinase activity of the complex

3. Most Cdks usually have at least one Cyclin partner

4. In humans there are at least 21 genes encoding Cdks however only about half of the Cdks are sufficiently studied = relatively well studied Cdks Human cell has 21 Cdks and 29 Cyclins

5. The Cdk complexes regulate various processes in cells Major functions: -Regulation of Cell Cycle (Cdk1,2,4,6,7) -Regulation of Transcription (Cdk7,8,9,12) Other functions: - regulation of pre-mRNA processing (Cdk11, Cdk9) - regulation of neuronal cell differentiation (Cdk5) - likely more functions to be discovered

6. Cdk complexes regulate various processes in cells

7. Regulation of kinase activity of Cdk complexes-overview Activation of Cdk kinase activity: -Association of Cdk with various Cyclin subunits -Phosphorylation of threonine in the “T-loop” of Cdk -Degradation of Cdk inhibitor proteins by ubiqitination and proteolysis Inhibition of Cdk kinase activity: -Binding of Cdk inhibitor proteins to Cyc/Cdk complexes -Inhibitory phosphorylation of Cdk -Ubiqitination and degradation of Cyclins in proteasome -Binding of Cdk inhibitor proteins together with small nuclear RNA to Cyc/Cdk complex

8. Activation of Cdk kinase activity: -Association of Cdk with various Cyclin subunits -Phosphorylation of Threonine in the “T-loop” of Cdk T-loop blocks active site (active site=ATP binding site) T-loop moves out of the active siteP-T-loop improves binding of substrate

9. Inhibition of Cdk kinase activity: -Binding of Cdk inhibitor proteins to Cyc/Cdk complexes P27 binding distorts and binds into the active site of Cdk2 (for example inhibits G1/S-Cdk in G1 phase)

10. Activation of Cdk kinase activity: - Degradation of Cdk inhibitor proteins by ubiqitination and proteolysis Cell cycle-dependent phosphorylation of Cdk inhibitor is a “mark” for recognition by SCF ubiquitin ligase, ubiquitinylation and degradation, rendering Cyc/Cdk complex more active

11. Inhibition of Cdk kinase activity: -Inhibitory phosphorylation of Cdk

12. Inhibition of Cdk kinase activity: -Ubiquitination and degradation of Cyclin by proteasome Mitosis-dependent activation of APC ubiquitin ligase leads to ubiquitination of Cyclin and its degradation

13. Inhibition of Cdk kinase activity: -Binding of Cdk inhibitor proteins and 7SK small nuclear RNA (7SK snRNA ) to CycT/Cdk9 complex P-TEFb=Cdk9 The kinase activity of Cdk9 is inhibited by binding to several proteins and small nuclear RNA, 7SK snRNA

14. Regulation of Cell Cycle by Cdks

15. Cell Cycle Cell cycle leads to production of two genetically identical daughter cells

16. Major events of the cell cycle S-phase – DNA synthesis-duplication of the chromosomes M-phase – mitosis-pair of chromosomes segregated into the nuclei – cytokinesis- the cell divides into two identical cells

17. The cell cycle has four phases G1 and G2 phases-time delay to allow the growth of the cell -time to monitor external and internal conditions before commitment to onset of S and M phase

18. The control of the cell cycle-three major checkpoints Control of the cell cycle triggers essential processes such as DNA replication, mitosis and cytogenesis

19. Cell cycle control system depends on cyclically activated Cdks Cyclical changes (expression and degradation) in Cyclin protein levels result in cyclic assembly/disassembly and activation/inhibition of Cyc/Cdk complexes; this leads to phosphorylation/dephosphorylation of proteins that initiate and regulate cell cycle events Cyclin protein levels change, Cdk protein levels are constant

20. Major Cyclins and Cdks in Vertebrates and Yeast

21. Comparison of the yeast and mammalian cell cycle Yeast- cell cycle is directed by one Cdk-Cdk1 (cdc28) Mammals-several Cdks (classical model), Cdk1 is essential to drive cell cycle in the absence of other Cdk (mouse knock out model)

22. Evolution of cell cycle control

23. Cell cycle control system is a network of biochemical switches where Cyc/Cdk complexes play a major role Cyc/Cdk: Cell cycle phases: Event:

24. Activation of M-Cdk (cycB/cdk1) De-phosphorylation activates accumulated M-Cdk at the onset of mitosis End of G2

25. Mechanism of cell cycle arrest in G1 by DNA damage DNA damage causes transcription of p21, Cdk inhibitory protein, that inhibits G1-S- and S-Cdks, arresting the cell cycle in G1 phase

26. Deregulation of cell cycle and cancer Cells escape from the proper control of the cell cycle during cancer development: -Increase in expression and activity of proteins driving cell cycle regulators (Cdks) -Inactivation of inhibitors of Cdks

27. Regulation of transcription by Cdks

28. Transcriptional Cyc/Cdk complexes

29. Major differences between Transcription and Cell Cycle Cyc/Cdk complexes Trancription Cyc/Cdks complexes: 1)Cdk has usually only one Cyclin partner 2)Usually in multi-protein complexes 3)The Cyclin levels in cells do not oscilate (Cdks need to be constantly active for basal transcription) 4)Regulated at the level of recruitment to specific gene

30. Ad 4) Examples of recruitment of P-TEFb (Cdk9) to genes

31. Differences between Cell Cycle and Transcription Cyc/Cdks-structure Sparse number of contacts btw Cyc and Cdk in transcription Cyc/Cdk complexes More contacts in Cell Cycle Cyc/Cdk complexes - important for Cdk activation

32. Differences between Cell Cycle and Transcription Cyc/Cdks- Cyclin structure All Cyclins have 2 canonical cyclin-boxes responsible for Cdk binding Each cyclin-box consists of 5 helixes The cyclin-boxes conserved in all Cyclins Cell Cycle and Transcription Cyclins differ significantly in sequence and structure outside of the cyclin boxes (binding to other proteins)

33. Differences between Cell Cycle and Transcription Cyc/Cdks- Cyclin structure

34. Comparison of Cdk9 and Cdk2 Structures very similar, sequence similarity 40% Cdk9 (green) /Cdk2 (orange) T-loop (T186/T180)

35. Transcription Transcription- synthesis of RNA from DNA template

36. Transcription in eukaryotes is tightly linked to co- transcriptional mRNA processing The co-transcriptional mRNA processing (capping, splicing, 3` prime end processing)

37. Transcription of protein-coding genes by RNA polymerase II (RNAPII) Promoter RNAPII Initiation Elongation Termination AAAA mRNA Gene Promoter RNAPII Pre-initiating RNAPII CTD C-terminal domain (CTD) of RNAPII plays a crucial role in regulation of transcription and co-transcriptional mRNA-processing Pre-mRNA

38. CTD consists of 52 repeats of heptapeptide YSPTSPS in which individual amino acids get phosphorylated to form a “CTD code” (Y 1 -S 2 -P 3 -T 4 -S 5 -P 6 -S 7 ) x52 P P P P P RNAPII CTD iso -52 repeats in humans (21 consensus, 31 non-consensus) -26 repeats in yeast -evolutionary conserved-important!

39. Human “CTD code” RNAPII

40. Repeats of the CTD get phosphorylated by the Cdks (Y 1 -S 2 -P 3 -T 4 -S 5 -P 6 -S 7 ) x52 Cdk9 Cdk7 P P P P P iso Cdk9 phosphorylates Serine (Ser) in the position 2 Cdk7 phosphorylates Serine (Ser) in the position 5

41. For the regulation of transcription cycle the phosphorylations of the CTD by the Cyc/Cdks are essential Promoter RNAPII GTFs Cdk7 Promoter RNAPII GTFs RNAPII Cdk9 RNAPII Pre-initiating RNAPII Initiated RNAPII Elongation Termination Ser2P AAAA Ser5P Mediator Capping enzyme Splicing/Chromatin remodeling Cleavage/PolyA factors factors

42. Modified CTD is a binding platform for transcription factors, RNA-processing factors and histone modification factors (code readers) RNAPII CTD Transcription factors RNA-processing factors Histone modification factors Exon Pre-mRNA Histones Phosphorylation of the CTD mediates: Transcription mRNA-processing Chromatin modifications RNA export Transcription-coupled genome stability

43. CTD code readers

44. Distribution of phosphorylated Serine 5 and Serine 2 in the CTD of RNAPII along the human protein coding genes Cdk7 (initiation) Cdk9 (elongation) RNAPII (Total) RNAPII Ser5-P RNAPII Ser2-P gene

45. Roles of new Cdks in the CTD modification (CTD code) (Y 1 -S 2 -P 3 -T 4 -S 5 -P 6 -S 7 ) x52 Cdk9 Cdk7 P P P P P iso Cdk12Cdk13 ? Cdk7Cdk9 ?

46. Cdks and their roles in transcriptional cycle of yeast and human

47. Deregulation of transcription by Cdks leads to the onset of human diseases -Cancer - aberrant kinase activity of Cdk9, Cdk12 defective transcriptional elongation, mRNA processing -HIV transcription- HIV Tat protein “steals” Cdk9 from its cellular complex to transcribe HIV genome

48. Cdk9 is recruited to most of RNAPII promoters and is present in catalytically active (small) and inactive (large) complexes and regulates transcriptional elongation MEPCE LARP7

49. Cdk9-dependent transcriptional elongation is a highly regulated process and its deregulation can lead to the onset of cancer HOX genes MYC gene Cdk9 Brd4 2- Mixed Lineage Leukemia (MLL) Abnormal fusion of MLL protein with Cdk9-containing complexes leads to aberrant elongation of Hox genes in leukemic cells Acute Myeloid Leukemia (AML) Expression of Myc gene regulated at the level of Cdk9-dependent transcriptional elongation in this Myc-dependent cancer.

50. Cdk12 is one of the most often mutated genes in ovarian carcinoma KD=kinase domain The mutations probably lead to the aberrant kinase activity and defective transcriptional elongation and/or mRNA processing of certain genes Cdk12 proposed to be a novel tumor suppressor W719 del E928 fs R882L E901C G909R K975E L996L T1014 del

51. HIV transcription is dependent on the Cdk9 (P-TEFb) protein HIV Tat protein “steals” Cdk9 from its complex with inhibitory Hexim1/7SK snRNA; resulting Tat/Cdk9 complex binds to HIV -TAR RNA element and drives HIV transcription in human cells